Cryo-EM structure of the bacterial divisome core complex and antibiotic target FtsWIQBL

In most bacteria, cell division relies on the synthesis of new cell wall material by the multiprotein divisome complex. Thus, at the core of the divisome are the transglycosylase FtsW, that synthesises peptidoglycan strands from its substrate Lipid II and the transpeptidase FtsI that crosslinks these strands to form a mesh, shaping and protecting the bacterial cell. The FtsQ-FtsB-FtsL trimeric complex interacts with the FtsWI complex and is involved in regulating its enzymatic activities, however, the structure of this pentameric complex is unknown. Here, we present the cryo-EM structure of the FtsWIQBL complex from Pseudomonas aeruginosa at 3.7 Å resolution. Our work reveals intricate structural details, including an extended coiled coil formed by FtsL and FtsB and the periplasmic interaction site between FtsL and FtsI. Our structure explains the consequences of previously reported mutations and we postulate a possible activation mechanism involving a large conformational change in the periplasmic domain. Since FtsWIQBL is central to the divisome, our structure is foundational for the design of future experiments elucidating the precise mechanism of bacterial cell division, an important antibiotic target.


General architecture of the FtsWIQBL complex
All five proteins were resolved in the final cryo-EM reconstruction (Fig. 1e), with FtsQ being partially disordered. The density for the membrane domain of PaFtsWIQBL reveals 13 transmembrane (TM) helices, including ten helices from FtsW, plus one each from FtsI, FtsB and FtsL (Extended Data Fig. 4a). Density for the FtsQ transmembrane helix (FtsQ™) was not observed (Extended Data Fig. 4b). The detergent micelle density was subtracted from the final reconstruction and the position of the complex in the membrane was approximated using the Orientations of Proteins in Membranes (OMP) webserver 9 ( Fig.  1e and Extended Data Fig. 4b, c).
The periplasmic domains of the PaFtsWIQBL complex extend about 70 Å away from the membrane in a "Y"-shape, with the FtsI transpeptidase domain (FtsI TP ) and the FtsQ β-domain (FtsQ β ) located on opposite arms of the Y, and FtsBL connecting them (Fig. 1e, f).
Interestingly, only FtsQ β is well-resolved, while density for the FtsQ polypeptide-transportassociated domain (FtsQ POTRA ) is only visible in low-resolution maps at high contour level, and density for FtsQ™ is completely absent (Extended Data Fig. 4d). FtsQ POTRA adopts a slightly different orientation relative to FtsQ β compared to previously determined X-ray structures [10][11][12] (Extended Data Fig. 4d). Taken together, this shows that FtsQ is tethered to FtsWILB via its FtsQ β -FtsB interaction, while FtsQ POTRA and FtsQ™ are flexibly attached in the current complex (Fig. 1f). As FtsQ™ is not visible, we assume it is not in the micelle that contains the other TM segments but might be surrounded by detergent molecules separately. While it has been previously reported that FtsB dimerises and could thus facilitate the dimerisation of core divisome components 13,14 , we find no evidence for higher oligomeric species in our cryo-EM data. We cannot exclude dimerisation of FtsB on its own, but in our current structure dimerisation via FtsB would be hindered by the presence of FtsI and/or FtsQ.
FtsL and FtsB have similar folds, each consisting of a long α-helical coiled coil segment, followed by a short α-helix and a β-strand. Interestingly, the FtsB α-helical coiled coil is interrupted by a small, conserved loop just above FtsB™ that might aid with sterically maintaining the correct insertion depth in the membrane (Fig. 1e, 2a). The observed interruption of the FtsB coiled coil has been postulated previously using computational models 14,15 . FtsB and FtsL interact with each other over their entire lengths through mainly hydrophobic interactions, e.g. between FtsL α1 and FtsB α2 (Fig. 2a and Extended Data Fig.  5a). Thus, our structure clarifies the FtsL-FtsB interaction and confirms previous reports that suggested a coiled-coil interaction between FtsB and FtsL 16,17 .
The transglycosylase FtsW and transpeptidase FtsI share two interfaces. The first interface is in the membrane, where FtsI™ interacts with TM8 and TM9 of FtsW (Extended Data Fig. 4a) -an interaction that closely resembles that of the previously reported RodA-PBP2 elongasome complex from Thermus thermophilus 18 . The second interaction site is located FtsI pedestal . Due to the flexibility of FtsI in this region, not all contacts could be determined unambiguously.
It has been previously reported that the cytoplasmic tail of E. coli FtsL is required for the recruitment of FtsW 19 . In the structure presented here, the FtsL cytoplasmic tail could not be traced unambiguously. This could either point towards a transient interaction during recruitment or species-specific differences in the recruitment due to the size of the cytoplasmic tail (11 residues in P. aeruginosa vs. 34 residues in E. coli). However, we clearly observe FtsL-FtsW interactions in the periplasm (FtsW EC1 and FtsL α1 ), and within the membrane through FtsW TM1 and the upper three turns of FtsL™. In the latter, the lower part of FtsL™ twists away from FtsW, due to its gyrating coiled-coil interaction with FtsB (Extended Data Fig. 5b).
Only a few FtsL-FtsQ contacts are present, however FtsL completes an extended β-sheet formed between FtsQ β strands β5 to β12 and FtsB β1 , by contributing its last β-strand (Fig.  1f, Fig. 2b panel 3). The FtsB-FtsQ interaction recapitulates that of previously determined crystal structures, where only small parts of FtsB 11,12 were resolved (Extended Data Fig.  6b). In addition, the cryo-EM structure shows an interaction between FtsB α2 (starting from E53) and FtsQ β loops (R183, S212-R214, R231). Interruption of the FtsB-Q interface with inhibitors based on the minimal interface could be expected to also disrupt the interface in the context of the divisome core complex 21 .

Comparison with RodA-PBP2 structures and AF2 predictions
Cell elongation in rod-shaped bacteria is facilitated by the elongasome that, like the divisome, polymerises and crosslinks PG, but is positioned throughout the cell envelope by moving MreB filaments 22 . RodA, the elongasome's transglycosylase is related to FtsW and has previously been structurally characterised using X-ray crystallography both on its own and as a RodA-PBP2 complex 18,23 (the latter being homologous to FtsWI). The structures of PaFtsW determined here and TtRodA are very similar, with the exception of TM7, which appears to be somewhat flexible in the cryo-EM structure, straighter with respect to that of TtRodA and closer to TM5 than in TtRodA-PBP2 (Extended Data Fig. 6c). It has been postulated that the movement of TM7 could open a cavity for the binding of the lipid tail of Lipid II to RodA 18 and the location of TM7 in PaFtsWIQBL creates such a cavity. The putative catalytic residue D275A is located in a deep, highly conserved cleft, as shown in Extended Data Fig. 6d, that we suggest might harbour the sugar moieties of Lipid II during the transglycosylase reaction.
The most striking observation when comparing PaFtsWIQBL with TtRodA-Pbp2 is the difference in the relative orientations of the TP with respect to the TG domain, despite the fact that the structures of the single proteins superimpose well on their own. Alignment of both complexes on the FtsW/RodA subunits places PaFtsI TP and TtPBP2 TP almost opposite to each other, requiring a ~130° rotation of PaFtsI TP /TtPBP2 TP for their interconversion (Extended Data Fig. 7a, b). The reason for this large difference is unclear, but is possibly caused by the presence of FtsQBL in our divisome structure and the absence of binding partners such as MreCD in the elongasome structure. Alternatively, the differences could be intrinsic to the elongasome and divisome complexes or reflect different, distinct states in the regulatory/catalytic cycle of the enzyme complexes.
We used AlphaFold 2 multimer 24 (AF2) to predict PaFtsWIQBL and many large-scale and fine features observed in the PaFtsWIQBL structure were predicted correctly by AF2, including the lack of an interaction between the membrane-embedded FtsQ™ and FtsWIBL™. However, in the AF2 model the periplasmic FtsIQBL interaction site is rotated upwards by about 30°, moving FtsI TP closer towards where the peptidoglycan layer is located (Extended Data Fig. 7a, c). Furthermore, a small rearrangement in the anchor subdomain FtsI pedestal -FtsL α1 shifts the interacting residues on FtsI pedestal from 208-212 to 203-206. To understand the implications of these differences, both structures were fitted into a to-scale model of the cell envelope of E. coli, produced from a cellular electron cryo-tomogram (Fig. 3a). Using the cryo-EM structure, the active site of FtsI TP does not reach the peptidoglycan layer, but does so in the more extended AF2 model. Since AF2 uses evolutionary couplings between amino acids 24 , in addition to protein structural features that correlate with sequence, it more likely predicts the active state of FtsWIQBL that one would expect to be selected for during evolution. Thus, the cryo-EM structure and AF2 prediction may represent the inactive and active (catalytic) states of the divisome core complex, respectively. Since our sample shows transglycoylase activity in vitro and the cryo-EM structure is substrate-free (apo), it is at least theoretically possible that Lipid II substrate binding contributes to the interconversion of the two states. In addition, other factors such as FtsN or peptidoglycan chains might be required to achieve the proposed conformational change. Recently, a study on RodA-PBP2 reported a similar upswinging mechanism 25 , which indicates that the concept of regulating the enzymatic activities via restricting access to the PG layer might be conserved between the divisome and the elongasome. However, significant differences exist between divisome and elongasome regarding the conformation before the upswinging motion and most likely also between the signals required to initiate this conformational change.
FtsN has been reported to trigger constriction in cells 26,27 . It is the last protein to be recruited to the division site in E. coli and its recruitment is dependent on the presence of earlier divisome proteins, including FtsA, FtsQ and FtsI 26,[28][29][30] . However, we have not been able to generate a biochemically stable E. coli FtsN-FtsWIQLB complex and previous studies reported that the addition of the FtsN periplasmic domain did not yield an increase in P. aeruginosa TG activity in vitro 7 . Whether FtsN activates the core divisome beyond the TG activity levels seen here through binding of FtsQLB, or whether the in vitro sample cannot be further activated will need further investigations including addition of other divisome components, e.g. FtsA, FtsN and/or DedD, as well as the substrate Lipid II. It is also possible that FtsN is involved only in regulating TP activity.

Interactions that affect divisome regulation
The Constriction Control Domain (CCD) of the divisome was identified previously from a set of mutations that allow partial or complete bypass of the requirement for FtsN in E. coli 31,32 . In our structure, these residues cluster at the top of FtsL α1 and FtsB α2 , with the FtsB CCD mutations facing FtsQ β . Furthermore, in close proximity are the Activation of FtsWI (AWI) residues on FtsL α1 that display a dominant-negative phenotype when mutated 19 (Fig. 3b). The CCD residues FtsB E61 (EcE56G/A/K/V/H) and FtsL Q65 (EcE88K/V) point towards a positively charged cavity formed by three arginine residues (FtsQ R214 , FtsQ R231 , FtsB R75 ), flanked by FtsL T69 (EcG92D, CCD residue) on one side and FtsB E64 (EcD59V, CCD residue) and FtsB T71 on the other side. This interface contains many charged and conserved residues (Fig. 3c), and removal of a charge or introduction of the opposite charge could well result in destabilisation of the interface and potentially increased flexibility of the protein. This may allow FtsWIQBL to more readily adopt an elongated, a more active conformation, as possibly indicated by the AF2 model, and with less or no activation signal, for example from FtsN.
A dominant negative phenotype was previously reported for the AWI mutation EcL86F 19 and its P. aeruginosa equivalent FtsL L63 interacts with FtsI F154 in FtsI pedestal (Fig. 3c).
Replacing the leucine with the bulkier phenylalanine likely causes a steric clash that weakens the FtsI-FtsL interaction. FtsL S66 (EcN89S, PaS66D) was classified as a CCD mutation in E. coli 31 , but has a dominant negative phenotype in P. aeruginosa, with reduced in vitro TG activity 7 , likely due to a steric clash with FtsI F154 . Mutation of FtsL L61 (EcL84K/D) has a dominant-negative phenotype and affects FtsL localisation to the septum 19,33 , indicating that the integrity of the FtsBL coiled-coil interaction is vital for a functional complex, as is well supported by our structure where the FtsBL coiled coil is at the centre of the complex.
The AWI residue FtsL R38 (EcR61C) is highly conserved and located between FtsW M257-I263 and the anchor subdomain of FtsI pedestal (Fig. 3d). Mutation of this residue causes a dominant-negative phenotype in both E. coli and P. aeruginosa and reduced in vitro TG activity in P. aeruginosa 7,19 . FtsL R38 might interact with the highly conserved FtsW G260 and FtsW S262 residues and stabilise FtsW M257-I263 together with FtsB R23 , a hypothesis supported by the fact that the corresponding loop in RodA and RodA-PBP2 is disordered 18,23 . The aforementioned FtsB coiled-coil discontinuity, C-terminal of FtsB R23 , might be required to allow for some flexibility of FtsB during the catalytic cycle of FtsW. EcFtsI L62P and TtPBP2 L43R correspond to PaFtsI V53 and result in a strong cell division defect and reduced TG activity in vitro, respectively 18,34 . PaFtsI V53 interacts with PaFtsW I257 in our structure, bringing the linker between FtsI™ and FtsI pedestal in close proximity to FtsW. Thus, FtsW M257-I263 presents an interaction site for FtsL, FtsB and FtsI in close proximity to the putative FtsW active site.
The recruitment of FtsQ to midcell requires FtsK 10,29,35 . AF2 predicts that the interaction between FtsK 1-222 and FtsWIQBL, occurs through FtsQ POTRA β2 and α3; this interaction is also identified by coevolutionary coupling analysis using EVcouplings 36 as an FtsQ-FtsK interaction hotspot (6/10 couplings, Extended Data Fig. 8a, b). Residues previously identified as impairing FtsK recruitment when mutated 10 (EcQ108, EcV92, EcV111, EcK113) map onto this conserved region (Extended Data Fig. 8c). We copurified an FtsQK 1-222 complex using E. coli proteins, which confirms that the two proteins interact tightly and constitutively (Extended Data Fig. 8d). To further our understanding of divisome recruitment and regulation, in the future larger divisome complexes will need to be assembled. For example, divisome interactions with FtsA, through FtsN, FtsK and possibly FtsQ have the potential to modify the conformations, oligomeric states and activities of the core complex and its enzymes.
We report the structure of the essential bacterial cell division complex and important future antibiotic target FtsWIQBL from Pseudomonas aeruginosa and show that PaFtsWIQBL forms a stable Y-shaped complex that harbours intrinsic TG activity. Our PaFtsWIQBL structure is able to explain many subunit contacts that have previously been shown to be important through loss-of-function and bypass mutations. In addition, an AF2 model reveals a different, likely catalytically competent state that allows for peptidoglycan crosslinking by FtsI. While our analysis hints at the nature of the catalytic state, further research is needed to resolve more states and their associated conformation changes, which possibly requires the addition of activating proteins such as FtsA and/or FtsN as well as the substrate Lipid II and its derivatives or products as ligands. It will be particularly exciting to resolve the enzymatic mechanism of the FtsW TG since it is a promising drug target for novel antibiotics given its function, conservation, periplasmic accessibility and very wide phylogenetic distribution. To gain a deeper understanding of the mechanism of the divisome, the inclusion of upstream and downstream proteins e.g., FtsEX, FtsK, PBP1b, and DamX will also be necessary. Our work is an important milestone in the 25-year quest for a molecular understanding of the ancient, near ubiquitous and medically important process of FtsZ-based bacterial cell division.

Cloning
Primers used for Gibson assembly are listed in Supplementary Table S2. The generated expression plasmids are listed in Supplementary Table S3. All constructs were confirmed by sequencing.

Baculovirus generation
Baculoviruses containing pFE758 and pNJ069 were used for insect cell expression of EcFtsWIQLB and EcFtsQK 1-222 respectively. Recombinant baculoviral genomes were generated by TN7 transposition in DH10bacY cells 39 and this bacmid was used to transfect Sf9 cells (Thermo Scientific) using FuGENE (Promega). After 3-5 days, the culture was centrifuged and the virus-containing supernatant was harvested and stored in the presence of 1% fetal bovine serum (FBS).

Bacterial expression
PaFtsWIQLB was expressed in E. coli. pLK1, pLK2 and pLK3 were sequentially transformed into E. coli C43(DE3). 120 mL of overnight culture were added to 12 L TB media with 2 mM MgCl 2 , kanamycin (25 µg/mL), chloramphenicol (25 µg/mL) and ampicillin (50 µg/mL), and grown at 37°C to an OD 600 of 0.7. Protein expression was induced with 1 mM IPTG and 1 g arabinose/L and continued at 18°C overnight. Cells were harvested by centrifugation for 20 min at 4,000 g at 4°C, flash frozen in liquid nitrogen and stored at -80°C.

Insect cell expression of EcFtsWIQBL and EcFtsQK 1-222
Sf9 cells were grown in Insect-Xpress medium (Lonza) to a density of 1.5-2 million cells/ml, infected with ~1% amplified baculovirus and harvested by centrifugation after Käshammer  EcFtsWIQBL reconstitution in nanodiscs for cryo-EM-The nanoquick protocol 40 was adapted to reconstitute the complex into nanodiscs. Proteins were expressed and the membrane was solubilised as described above but instead of GDN a mixture of LMNG:CHS (10:1) was used at a final concentration of 1%. The solubilised protein was incubated overnight with StrepTactin sepharose beads (Cytiva). The beads were washed in Strep Buffer including 0.1% LMNG:CHS (10:1) and then into SEC buffer including 2% glycerol and 0.1 % LMNG:CHS (10:1). A thousand fold molar excess of POPG was added and incubated for an hour, followed by a 20-fold excess of MSP2N2 (purified as described in 41 ). After 15 min, activated SM-2 BioBeads (BioRad) were added and the mixture was rotated overnight. The protein complex was eluted in Strep Buffer supplemented with 2.5 mM desthiobiotin (Sigma) and subjected to size-exclusion chromatography as above in SEC buffer without detergent. .0, 300 mM NaCl). Monomer peak fractions were combined and concentrated to 52 g/L by centrifugal filtration (Vivaspin), then flash frozen in aliquots before being stored at -80°C.

Cryo-EM single particle structure determination
Grid preparation PaFtsWIQBL-Grids were prepared with freshly purified protein from the peak fraction of the SEC elution at a final concentration of 1. Data processing-Unless stated otherwise, all processing was done in RELION 4.0 43 . Motion correction was performed using RELION's own implementation of the MotionCor2 algorithm 44 with 5x5 patches. CTFFind-4.1 45 was used for CTF estimation. The initial reference was generated in CryoSPARC 46 from a different dataset of the same sample (not used for the final reconstruction). Particles were picked with Topaz 47 7,276,623 particles total, (1,398 per micrograph on average) and extracted 4x binned with a boxsize of 70 px and a pixel size of 4.36 Å/px. The particles were split into seven subsets for initial 3D classification into three classes. The best classes of each job were selected and refined. The refined particles were combined into two sets of particles and subjected to 3D classification without alignment. The best classes from each alignment were selected (1,997,326 and 2,922,553 particles total), combined and subjected to a 3D refinement. A mask (extended by 2 px and added soft edge of 2 px) was used to subtract the micelle density from the complex. The subtracted particles were subjected to a 3D classification without alignment and two classes were selected (344,300 and 461,499 particles), re-extracted (2x binned with a boxsize of 140 pix and a pixel size of 2.18 Å/px) and subjected to 3D refinement. 3D classification without alignment was run and the best class (160,615 particles) was selected. The particles were re-extracted (no binning, 280 pix, 1.09 Å/px) and subjected to one round of polishing, 3D refinement, CTF refinement and 3D refinement. Following subtraction of the micelle the particles were subjected to a round of 3D refinement and 3D classification without alignments. From this 3D classification, the best class was selected (136,364 particles) and 3D refined two times, the second time using a mask that excluded the POTRA domain of FtsQ. After postprocessing with the calibrated pixel size (see below) the final reconstruction had an overall resolution of 3.7 Å as determined by Fourier shell correlation (FSC, cutoff 0.143).
Model building-The pixel size was calibrated using Chimera 48

Lipid II extraction
Lipid II was extracted from Enterococcus faecalis (DSMZ 2570) as described before 54 .
Briefly, an overnight culture of E. faecalis was diluted 1:100 into 3 L of Brain heart infusion (BHI, Merck) and grown at 37°C, 180 rpm to an OD 600 of 0.7. Vancomycin (Sigma) and moenomycin (Santa Cruz) were added at 10 µg/mL and 5 µg/mL, respectively, and after 20 min cells were centrifuged in pre-cooled bottles at 4,500 g for 20 min. Cell pellets were resuspended in BHI, centrifuged in Falcon tubes at 3,200 g for 10 min, flash frozen and stored at -20°C overnight. Frozen cell pellets were thawed in a total of 30 mL phosphate-buffered saline (PBS), divided equally into two 250 mL glass Erlenmeyer flasks and 17.5 mL chloroform and 35 mL methanol were added. After 2 h of vigorously stirring at room temperature, the mixture was spun in Teflon tubes for 10 min at 4,000 g at 4°C and the supernatant from each Erlenmeyer was combined with 30 mL chloroform and 22.5 mL PBS. The mixture was stirred vigorously for 2 h at room temperature, then spun for 10 min at 4,000 g at 4°C. The tubes were left at room temperature for 1 h until the supernatant was clear. The interface was then transferred with a glass Pasteur pipette to a 25 mL separatory funnel and left to settle overnight at 4°C. The lower organic phase was discarded and the interface dried in a 25 mL round bottom flask on a rotary evaporator at 40°C. The dried interface was resuspended in 7.5 mL pyridinium acetate:n-butanol (1:2) (PB) and 7.5 mL n-butanol-saturated water. Pyridinium acetate was prepared by adding 51.5 mL glacial acetic acid dropwise to 48.5 mL pyridine and filtered before use. The Lipid II extract was transferred to a 25 mL separatory funnel and the bottom phase was re-extracted with 5 mL PB. The top phase from the re-extraction was added to the top phase in the separatory funnel and extracted three times with 5 mL n-butanol-saturated water. The top phase was dried using a rotary evaporator at 40°C and resuspended in chloroform:methanol (1:1), partially dried under a stream of nitrogen gas and then transferred to a 250 µL non-stick glass vial (Agilent), in which it was dried completely. This was repeated 4 times to ensure efficient transfer before resuspending the Lipid II extract in 210 µL chloroform:methanol (1:1). The Lipid II extract was assessed by spotting 1-2 µL on a HPTLC silica gel 60F254 plate (Merck). The TLC plate was developed in a mixture of chloroform:methanol:water:ammonia (88:48:10:1) and Lipid II was visualised by heating the plate after soaking in phosphomolybdic acid (PMA) as described previously 55 .

Deprotection of FMOC-BDL
D-Lys-Biotin (BDL) was prepared following a standard deprotection protocol 42 . Briefly, 15 mg Fmoc-D-Lys(Biotin)-OH (Santa Cruz Biotechnology) was stirred in 3.1 mL of 20% piperidine/dimethylformamide and 466 µL toluene for 40 min at room temperature, then dried in vacuum at 50°C. The sample was resuspended in 5 mL water, stirred for 2 h at room temperature and then filtered through a 0.22 µm filter. The filtrate was pipetted into a tared tube, frozen on dry ice and then freeze dried. The residue was dissolved in water to make a 10 mM stock, aliquoted and stored at -20°C.

Transglycosylase activity assay
Lipid II used to monitor glycosyltransferase activity of the protein complex was dried using a nitrogen stream and dissolved in an equal volume of dimethyl sulfoxide (DMSO). The reaction and detection of glycan strands was adopted from previously published protocols 3,23,42,56

Electron cryo-tomography of E. coli cells
A culture of E. coli strain B/r H266 expressing plasmid pRBJ212 57 was grown in LB media at 37°C, 180 rpm to an OD 600 of 0.6. Cells were concentrated 10x by centrifugation and mixed with 10 nm protein A gold fiducials in a 1:10 ratio. 4 µL of this mixture was applied to a 200-mesh Cu2/2 grid (Quantifoil), back-blotted and plunge-frozen in liquid ethane using a manual plunger. Cells were thinned by cryo-focused ion beam milling (cryoFIB) using a Scios dual beam FIB-SEM (TFS). Before milling, grids were sputtered with a protective layer of organic platinum using the gas injection system. Lamellae were milled in a stepwise fashion, gradually reducing the beam current as the lamellae were thinned, from 1 nA to 30 pA and at a nominal milling angle of 10°. CryoET was carried out on a Krios microscope (ThermoFisher) equipped with a Gatan imaging filter and K2 camera. Tilt series were collected using serialEM software 58 , using a bidirectional tilt scheme from -10° (to flatten the lamella) with a 2° increment and a total dose of 112 e -/Å 2 , divided over 56 images, each with 10 frames. The pixel size was 3.7 Å and the defocus target was -5 µm. Frame alignment and tilt series alignment were performed using IMOD 59 , 2x binned aligned tilt series were generated and used to generate a SIRT reconstruction using tomo3D 60 , which were then low-pass filtered to 20 Å. Fig. 4

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.  Grey boxes indicate regions that are further discussed in Extended Data Fig. 5a (ED5a). b) Key interactions within the FtsWIQBL periplasmic domain are highlighted in boxes 1 to 3. The grey box indicates a region that is further discussed in Extended Data Fig. 5b (ED5b). Panels 1-2: the interactions between FtsI pe destal and the FtsL coiled coil are shown in two panels. Most residues in the interface between FtsI and FtsL in this region are hydrophobic or neutral. Residues in FtsL that also face FtsB are highlighted in violet. Panel  shows a high degree of conservation for many residues in this area. d) Residues surrounding the region of discontinuity in the FtsB coiled coil. This region contains residues with known loss-of-function mutations (shown in bold). Many residues in this region are highly conserved, including FtsL R38 (R61 in E. coli), which inserts between FtsI and FtsW and is located close to a highly conserved loop in FtsW (M257-I263, Q279-V285 in E. coli) that is in close proximity to the putative active site residue D275 (D297 in E. coli).